Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein βγ subunit dimer (Gβγ). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gβγ expression and G protein signaling. This inhibition was caused by an inability of nascent Gβγ to form dimers. Phosphorylation of PhLP at serines 18–20 by protein kinase CK2 was required for Gβγ formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gβ in the absence of Gγ, and S18–20 phosphorylation was required for Gγ to associate with the PhLP-Gβ complex. Once Gγ bound, PhLP was released. These results suggest a mechanism for Gβγ assembly in which PhLP stabilizes the nascent Gβ polypeptide until Gγ can associate, resulting in membrane binding of Gβγ and release of PhLP to catalyze another round of assembly

Abstract. Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted a-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NHE-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srplcx) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.T RE nuclear pore complex (NPC), 1 a large supramolecular assembly spanning the nuclear envelope (NE), mediates molecular exchanges between the nucleus and cytoplasm. The NPC contains aqueous channels that accommodate the passive diffusion of molecules smaller than N20--40 kD, but transport of most macromolecules occurs by signal-mediated, energy-dependent mechanisms (reviewed by Fabre and Hurt, 1994;Pant6 and Aebi, 1994;Rout and Wente, 1994; GSrlich and Mattaj, 1996). Targeting signals for nuclear protein import, termed nuclear localization sequences (NLSs), are usually short stretches of amino acids enriched in basic residues that can occur as single or bipartite motifs (Kalderon et al., 1984;Robbins et al., 1991).

As olive oil is the main source of calories in the Mediterranean diet, a large number of studies have been carried out to characterize its role in various diseases and exploitation for the prevention and treatment of hypertension, carcinogenesis, diabetes, atherosclerosis, and other diseases. As one of the major polyphenols present in virgin olive oil, hydroxytyrosol shows a variety of pharmacological activities such as antioxidant properties, anticancer, anti-inflammatory, and neuroprotective activities, and beneficial effects on the cardiovascular system, which show its potentiality for the development of dietary supplements. In the future, more attention should be paid to its action mechanism in vivo and synergistic effect. Further research will be performed to provide the theoretical basis for hydroxytyrosol and its derivatives use as health supplements.

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein ␤␥ subunit complex (G␤␥) that has been recently shown to catalyze the formation of the G␤␥ dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for G␤␥ dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the G␤␥-assembly process. The results reported here delineate a mechanism for G␤␥ assembly in which a stable ternary complex is formed between PhLP, the nascent G␤ subunit, and CCT that does not include G␥. PhLP phosphorylation permits the release of a PhLP⅐G␤ intermediate from CCT, allowing G␥ to associate with G␤ in this intermediate complex. Subsequent interaction of G␤␥ with membranes releases PhLP for another round of assembly.Eukaryotic cells employ heterotrimeric G proteins to transduce a wide variety of hormonal, neuronal, and sensory signals that control numerous physiological processes. As a result, malfunctions in G protein pathways contribute to many diseases (1-3), and therapeutic agents targeting G protein-coupled receptors represent the single largest class of current pharmaceuticals (4). There are three fundamental steps in the propagation of a G protein-mediated signal. First, a ligand binds a receptor, resulting in a change in the packing of the seven transmembrane ␣-helices found in all G protein-coupled receptors. Second, the activated receptor catalyzes exchange of GDP for GTP on the ␣ subunit of a heterotrimeric G protein (G␣) 2 on the intracellular surface of the receptor. GTP binding causes G␣ to dissociate from the G protein ␤␥ subunit complex (G␤␥). Third, the G␣⅐GTP and G␤␥ complexes control the activity of effector enzymes and ion channels that regulate the intracellular concentration of second messengers (cyclic nucleotides, inositol phosphates, and Ca 2ϩ ) and the plasma membrane electrical potential (mainly via K ϩ channels). Changes in these properties in turn orchestrate the cellular response to the stimulus (5).Phosducin-like protein (PhLP) is a member of the phosducin gene family (6 -8) that is believed to participate in G protein signaling by virtue of its ability to bind the G␤␥ dimer with high affinity (9 -11). Many in vitro and overexpression experiments have shown that PhLP binding to G␤␥ blocks its ability to interact with G␣ or effectors (9,10,(12)(13)(14). From these experiments, it was suggested that the physiological role of PhLP was to down-regulate G protein signaling by sequestering G␤␥. However, the results of several recent studies have seriously challenged this model. Specifically, disruption of the PhLP1 gene in the chestnut blight fungus Cryphonectria parasitica (15) and in the soil amoeba Dictyostelium discoideum (7) yielded the same phenotype as the disruption of the G␤ gene. Moreover, PhLP deletion blocked G protein signaling in Dictyosteliu...

Lipids, which have a core function in energy storage, signalling and biofilm structures, play important roles in a variety of cellular processes because of the great diversity of their structural and physiochemical properties. Lipidomics is the large-scale profiling and quantification of biogenic lipid molecules, the comprehensive study of their pathways and the interpretation of their physiological significance based on analytical chemistry and statistical analysis. Lipidomics will not only provide insight into the physiological functions of lipid molecules but will also provide an approach to discovering important biomarkers for diagnosis or treatment of human diseases. Massspectrometry-based analytical techniques are currently the most widely used and most effective tools for lipid profiling and quantification. In this review, the field of massspectrometry-based lipidomics was discussed. Recent progress in all essential steps in lipidomics was carefully discussed in this review, including lipid extraction strategies, separation techniques and mass-spectrometry-based analytical and quantitative methods in lipidomics. We also focused on novel resolution strategies for difficult problems in determining C=C bond positions in lipidomics. Finally, new technologies that were developed in recent years including single-cell lipidomics, flux-based lipidomics and multiomics technologies were also reviewed.